Method of Methane Conversion Using Aerobic Methanotrophs

This application claims the benefit and priority to Korean Patent Application No. 10-2024-0058464, filed on May 2, 2024. The entire disclosure of the application identified in this paragraph is incorporated herein by references.

This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing XML file entitled “000387 us_SequenceListing.XML”, file size 14,901 bytes, created on 7 Jan. 2025. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

The present disclosure was carried out under the support of the Ministry of Science and ICT, with the unique project number 1055001131, sub-project number 2015M3D3A1A01064929. The research management agency for this project is the National Research Foundation of Korea, and the project is titled “development of climate change mitigation technologies” with the research task named “Infrastructure research of C1 gas conversion technologies” The leading institution is Sogang University, and the research period spans from Jan. 1, 2023, to Dec. 31, 2023.

The present disclosure relates to technology for Methane Capture, Utilization, and Sequestration (MCUS) using aerobic methanotrophs. More specifically, the present disclosure pertains to a cultivation method that periodically supplies pure Oto easily separate carbon dioxide generated during methane oxidation without an additional COcapture process and simultaneously obtain high-value products generated from aerobic methanotrophs, as well as a process applying the method.

If the Earth's temperature rises by more than 2° C., natural disasters such as heatwaves, cold waves, heavy snowfall, typhoons, and wildfires occur. Therefore, with the goal of limiting the Earth's temperature rise to less than 1.5° C., major countries around the world have begun to participate in “carbon neutrality,” aiming to reduce greenhouse gas emissions, which cause abnormal weather, to “zero.” The EU, Germany, the UK, Japan, and others have declared carbon neutrality by 2050-2060, and South Korea is also taking active steps to achieve carbon neutrality by establishing a 2050 carbon neutrality scenario.

To achieve this goal, the development of various COreduction technologies, such as process electrification, COCapture and Utilization (CCU), and renewable energy generation, is actively progressing. Recently, as industry interest in methane emissions, in addition to carbon dioxide, has increased, the “Global Methane Pledge” was declared. Methane is one of the six major greenhouse gases defined by the Kyoto Protocol and has a global warming potential 21 times higher than carbon dioxide. Although there are technologies to convert methane into liquid fuel through chemical methods to reduce methane emissions, these methods have drawbacks. They are less economically and environmentally efficient than existing methane utilization processes due to extreme reaction conditions such as high temperatures and high pressures and the thermodynamic limitations caused by gas-phase equilibrium. Therefore, the biological conversion of methane using methanotrophs is relatively simple and can operate at room temperature and pressure. It can be proposed as an alternative that combines the reduction of methane, which would otherwise be burned and released into the atmosphere, with the production of value-added products.

Methane conversion technology using methanotrophs has been studied for various products such as organic acids, alcohols, and secondary metabolites, and shares a commonality with existing oxidative methane conversion in terms of the conversion under aerobic conditions, which requires oxygen. Aerobic methanotrophs can be divided into three main groups based on various assimilation pathways: Group I (ribulose monophosphate pathway (RuMP); Type I, Type X), Group II (serine pathway; Type II, Type III), and group III (Calvin-Benson-Bassham pathway; Type IV). Most studies on biological methane conversion have focused on methanotrophs such as Type I and Type II, which primarily produce high-value chemicals such as polyhydroxyalkanoates, single-cell proteins, methanol, organic acids, and ectoine. Despite numerous cultivation studies using methanotrophs, efforts to scale this technology to commercial-scale processes have been limited by low gas-liquid mass transfer, low process productivity, and conversion rates. Therefore, strategies to maximize methane conversion rates and recover as many products as possible are necessary.

According to the stoichiometry of the reaction involving methanotrophs (aCH+bO→cCell+dProducts+eCO+fHO), it can be seen that carbon dioxide is inevitably generated during cell growth and chemical production. This contradicts the claim that the biobased methane conversion reaction is environmentally friendly. Therefore, to achieve the inherent goal of biological methane conversion by methanotrophs, which is to convert methane into environmentally friendly high-value products, it is crucial not only to increase methane conversion rates and product yields but also to eliminate carbon dioxide emissions generated during the reaction.

In existing methane fermentation processes using aerobic methanotrophs, methane and air are used for methane oxidation. When gases discharged from the reactor are recirculated to maximize methane conversion rates, the accumulation of nitrogen lowers the partial pressure of carbon dioxide and methane in the final gas products, making the use of CCU essential.

With the problems in mind, the present inventors have succeeded in using aerobic methanotrophs to produce high-value chemicals from methane while capturing the emitted carbon dioxide without the need for additional technologies such as CCU, leading to the present disclosure.

Thus, the present disclosure aims to provide a methane conversion method including the following steps: a cultivation step of periodically supplying oxygen to a reaction unit containing methane and oxygen to culture aerobic methanotrophs; and a final gas separation step of separating a final gas containing carbon dioxide from the products generated by the aerobic methanotrophs.

The present disclosure relates to technology for Methane Capture, Utilization, and Sequestration (MCUS) using aerobic methanotrophs. More specifically, the present disclosure pertains to a cultivation method that periodically supplies pure Oto easily separate carbon dioxide generated during methane oxidation without an additional COcapture process and simultaneously obtain high-value products generated from aerobic methanotrophs, as well as a process applying the method.

Below, a detailed description will be given of the present disclosure.

One aspect of the present disclosure is directed to a methane conversion method comprising the following steps: a cultivation step of periodically supplying oxygen to a reaction unit containing methane and oxygen to culture aerobic methanotrophs; and a final gas separation step of separating a final gas containing carbon dioxide from the products generated by the aerobic methanotrophs.

As used herein, the term “methanotroph” refers to a prokaryotic species that uses methane as a carbon source and chemical energy source for their life activities. Energy can be generated during the process of methane oxidation by methanotrophs, and methanol (CHOH) may be produced as a result of methane oxidation. Methanotrophs can be obligate aerobic, microaerobic, facultative anaerobic, or obligate anaerobic bacteria. For example, obligate aerobic or microaerobic methanotrophs can be used in the ectoine production process, but with no limitations thereto.

The methanotroph may be at least one selected from the group consisting ofspp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp.,spp., orspp., with no limitations thereto.

In an embodiment of the present disclosure, the methanotroph may belong to the genus

In an embodiment of the present disclosure, the methanotroph may be the strain20Z.

The strain20Z is registered with the strain number DSM 19304 at the German Collection of Microorganisms and Cell Cultures (DSMZ) and can be obtained by purchase or distribution from DSMZ.

In an embodiment of the present disclosure, the methanotroph may be a strain obtained by removing the endogenous plasmid and deleting all or part of the ectoine hydroxylase gene (ectD) or the ectoine biosynthesis regulatory gene (ectR) from20Z.

The term “endogenous plasmid,” as used herein, refers to a plasmid that a cell naturally possesses before any external manipulation is performed. Endogenous plasmids can be linear or circular and may include genes that positively or negatively affect the life activities of the cells containing the endogenous plasmid. For example, methanotrophs may possess doeA, ectD, or ectR genes on endogenous plasmids, in addition to those present in their genome.

The term “ectoine hydrolase gene” (doeA), as used herein, refers to a gene encoding ectoine hydrolase, which is used by halophilic or halotolerant microorganisms to degrade ectoine for a carbon and nitrogen source. The ectoine hydrolase expressed from doeA hydrolyzes ectoine to convert it into an amino ester, which then becomes the substrate for subsequent enzymes and ultimately gets broken down into amino acids. The gene doeA can also be referred to as eutD.

When doeA is deleted in methanotrophs, the methanotrophs cannot hydrolyze already produced ectoine, thus providing a foundation for increasing ectoine production through methanotrophs.

The term “ectoine hydroxylase gene (ectD),” as used herein, refers to a gene encoding ectoine hydroxylase, which adds a hydroxyl group (—OH) to ectoine with the consequent conversion into hydroxyectoine. The hydroxyectoine produced from ectoine by ectoine hydroxylase expressed from ectD can act as an osmoprotectant for the cell, similar to ectoine.

The term “ectoine biosynthesis regulatory gene” (ectR), as used herein, refers to a gene encoding a transcriptional repressor for the ectABC operon or the ectABC-ask operon, which encodes a series of enzymes that induce ectoine biosynthesis.

In an embodiment of the present disclosure, a suicide vector may be used to delete all or part of the genes doeA, ectD, and ectR included in the genome of methanotrophs.

The term “suicide vector,” as used herein, refers to a non-replicable vector carrying a suicide gene that induces the deletion of a target gene from the host cell chromosome through homologous recombination and counterselection.

Specifically, sequences of the upstream and downstream regions of the gene to be deleted are inserted into the suicide vector and introduced into the host cell to induce homologous recombination with the host cell's chromosome. After transformation, culturing the host cells in a medium containing a substance that induces counterselection allows for the selection of transformed cells in which the suicide vector homologously recombined into the chromosome has been excised from the chromosome through subsequent homologous recombination. consequently, the genome of the final selected host cells will have all or part of the target gene deleted, effectively eliminating the function of the target gene through the suicide vector.

For example, by recombining the sequences of the upstream and downstream regions of the doeA gene into a suicide vector, and then performing homologous recombination and counterselection with the host cell chromosome, the doeA gene can be deleted from the host cell's genome.

The term “suicide vector” can be used interchangeably with “suicide plasmid.”

In an embodiment of the present disclosure, the methanotroph may be the strain20ZDP3.

In an embodiment of the present disclosure, a strain of20Z strain from which the endogenous plasmid has been removed and in which all or part of ectD and ectR have been deleted, without deletion of all or part of doeA, may be referred to as the20ZDP2 strain. A strain from which all or part of doeA has additionally been deleted, along with the removal of the endogenous plasmid and all or part of ectD and ectR, may be referred to as the20ZDP3 strain.

The20ZDP2 strain and the20ZDP3 strain have been deposited with the Korea Research Institute of Bioscience and Biotechnology (KRIBB) under accession numbers KCTC19047P (International accession number: KCTC16115BP) and KCTC19076P (International accession number: KCTC16116BP), respectively.

The reaction unit may have a volume of 250,000 to 400,000 L, for example, 311, 119.4 L, but is not limited thereto.

The methane may be included at 50 to 100% by volume of the reaction unit, preferably at 50 to 90%, 60 to 100%, 60 to 90%, 70 to 100%, or 70 to 90% by volume, for example, at 90% by volume, but is not limited thereto.

In the cultivation step, oxygen may be supplied such that the partial pressure of oxygen in the reaction unit is 1 to 20% of the partial pressure of methane in the reaction unit, preferably 5 to 20%, 10 to 20%, or 15 to 20%, for example, 20%, but with no limitations thereto.

The supply cycle of oxygen may be determined based on the degree of oxygen consumption in the reactor, for example, when all the oxygen in the reactor is consumed. The supply cycle may be 7 to 12 hours depending on the process conditions, but is not limited thereto.

The reaction unit may comprise 2 to 10 reactors, but with no limitations thereto. By operating multiple batch reactors with a time lag, a continuous methane conversion process can be realized from the perspective of the entire reaction unit.

Therefore, the number of reactors can be adjusted according to the methane assimilation rate of the methanotrophs used in the process. When using methanotrophs with a fast methane assimilation rate, a continuous methane conversion process can be realized with a smaller number of reactors due to the faster methane conversion rate per reactor unit.

The oxygen supplied to the reaction unit may be pure O, without other components. Supplying pure oxygen instead of air to the reaction unit can prevent the accumulation of nitrogen in the reaction unit.

The cultivation step may further include a nitrogen source supply step that supplies a nitrogen source to the reaction unit, but with no limitations thereto.

The term “nitrogen source,” as used herein, refers to a substance that can provide nitrogen compounds to the cells. By adding a nitrogen source to the medium in which the cells are cultured, the cells can utilize the nitrogen compounds in the medium, thereby achieving higher growth rates of the methanotroph strain and increased production of the product.

The nitrogen source supply step may continuously supply nitrogen at a concentration of 1 to 10 g/L per 1 g/L of aerobic methanotroph, preferably at concentrations of 1 to 9 g/L, 1 to 8 g/L, 1 to 7 g/L, and 1 to 6 g/L, for example, at a concentration of 1 to 5 g/L, but with no limitations thereto.

The nitrogen source may be selected from one or more of the group consisting of potassium nitrate (KNO), sodium nitrate (NaNO), ammonium chloride (NHCl), ammonium sulfate ((NH)SO), yeast extract, urea, peptone, tryptone, or beef extract, but is not limited thereto.

The cultivation step may further include a step of continuously supplying an alkaline solution to adjust the pH of the reaction unit, but with no limitations thereto.

The alkaline solution may be at least one selected from the group consisting of NaOH, NHOH, or KOH, but is not limited thereto.

The product may include at least one from the group consisting of ectoine, methanol, PHA (polyhydroxyalkanoate), PHB (polyhydroxyvalerate), PHBV (poly(3-hydroxybutyrate-co-3-hydroxyvalerate)), microbial protein, biodiesel precursor, lactic acid, butyric acid, acetic acid, muconic acid, succinic acid, 3-hydroxypropionic acid, 2,3-butanediol, putrescine, cadaverine, and sesquiterpene, but is not limited thereto.

The term “capture,” as used herein, refers to the selective separation of a specific gas from a gaseous mixture. By culturing methanotrophs that use methane as the main energy source, methane in the reactor can be captured and converted into high-value products such as ectoine.

The stoichiometric equations for cell growth and ectoine production by methanotrophs can be represented as shown in Equations 1 to 3 below. Nitrogen is essential for all life activities of methanotrophs (Equation 1), and extra nitrogen is particularly required for ectoine biosynthesis (Equation 2). Thus, the addition of a nitrogen source can promote the ectoine biosynthesis by methanotrophs. This reaction can also be interpreted by adding the reaction of methane and oxygen, i.e., mineralization, which generates carbon dioxide (Equation 3).